Brown and colleagues (Nature 1993) cloned a novel calcium-sensing receptor (CaR) which is a member of the G protein-coupled receptor (GPCR) superfamily, specifically subfamily 3 which includes metabotropic glutamate, GABA-B, taste, and putative pheromone receptors. The CaR is expressed in a variety of cell types including kidney, brain, thyroid C cells, and most prominently parathyroid cells, and is involved in extracellular calcium homeostasis. The CaR cDNA predicts a 7 transmembrane (7TM) core typical of GPCR but with a large (approximately 600 residue) N-terminal extracellular domain (ECD). We are studying the receptor's structure and function in order to understand how calcium binding to the receptor leads to G protein activation. We have raised monoclonal antibodies to synthetic peptides corresponding to sequences in the ECD of the receptor. These antibodies have proved very useful in immunoblot and immunocytochemistry studies of the receptor. We have also succeeded in expressing and purifying the ECD,and in generating monoclonal antibodies against the purified ECD. These antibodies have interesting functional effects on the CaR, and are being evaluated for their epitopes to help define receptor structure/function. We found that the ECD is an intermolecular disulfide-linked dimer that accounts for the dimeric nature of the intact receptor. Mutagenesis of ECD cysteines has defined which are essential for receptor expression and has identified the cysteines (129 and 131) responsible for receptor dimerization. Disulfide mapping of the ECD using cleavage of the purified protein followed by mass spectrometry is in progress. We have characterized the functional effects of missense mutations identified in subjects with autosomal dominant hypocalcemia (ADH). Most such mutations cause increased sensitivity of the receptor to calcium. We have modeled the ECD structure based on its homology to bacterial periplasmic binding proteins known to have a bilobed, "venus flytrap" structure, and are testing this model using mutagenesis and biochemical approaches. We have shown that a putative loop in the ECD comprising residues ~115-139 forms the dimer interface and is critical for receptor activation. This loop is a "hotspot" for naturally occurring, activating mutations. We have also shown that a cysteine-rich region at the end of the ECD forms a separate domain that plays a critical role in linking calcium activation of the ECD venus flytrap domain to activation of the 7 transmembrane domain of the CaR. We have identified an acidic residue, E837, as critical for the action of both positive and negative allosteric modulators of the receptor. This residue is within a region at the junction of TM6 and TM7 that is another hotspot for activating mutations in subjects with ADH. We also showed that a negative allosteric modulator can normalize calcium sensitivity in cells transfected with activating mutant CaR, suggesting a more effective therapy for ADH. Future studies, including engineered mouse models expressing modified versions of the receptor and/or the G proteins to which it couples are directed at elucidating in detail the mechanism of receptor activation by calcium.